A range of analytical techniques employing optical means are available to provide information about substances of interest. Typically, the sample of interest is illuminated with light and, depending on the technique employed, the resulting emitted, scattered or transmitted light is collected, spectrally analysed as appropriate and then detected using some form of optical detector.
Optical analytical techniques in common use include fluorescence spectroscopy, optical absorption spectroscopy, infra-red absorption spectroscopy, light scattering and Raman spectroscopy.
Each form of optical analytical technique can provide different information about a substance of interest and it is therefore often advantageous to perform multiple different forms of optical analysis on a given sample in order to provide a deeper understanding of the sample's properties.
A range of containers are available to allow the performance of individual optical measurements from single liquid samples and these are generally described as ‘cuvettes’. Different designs are generally used for different optical measurement techniques although in principle a simple square cross section cuvette with four transparent walls could be used for multiple optical analytical techniques. Heating/cooling blocks are available to control the temperature of conventional cuvettes. Although cuvettes are available for a wide range of sample volumes, these sample volumes are generally larger than 50-100 μl, although special cuvettes are available that in favourable circumstances can use as little as 12 μl.
Widely used multi-container arrays are 96 well, 384 well and 1536 well ‘micro-titre’ plates conforming to standards published by the Society for Biomolecular Screening. These can be either viewed from the top or in some cases through the bottom of the well. These sample plates do not however allow illumination and collection of light at a wide range of angles and thus the optimum configuration for many of the optical analytical techniques can not be achieved. Although it is possible to perform a range of optical analytical techniques in these plates, therefore, the sample volumes generally have to be relatively large to compensate for the sub-optimal optical configuration.
A particular problem arises when optical absorption measurements are performed in standard micro-titre plates due to the optical path length being affected by both sample volume and meniscus affects which can lead to inaccurate measurements. While standard micro-titre plates can be heated and cooled their design makes the simultaneous application of multiple optical analytical techniques difficult. Real time polymerase chain reaction (RT-PCR) thermo cyclers represent the current state of the art in multi-well sample temperature control however these only allow visible fluorescence measurements and the lack of an optimised excitation-collection geometry requires that large sample volumes are required.
There are a wide range of spectrometers and light scattering instruments available that perform a single type of optical analysis on one sample at a time. Examples include fluorescence spectrometers exemplified by the RF-5301PC from Shimadzu, optical absorption spectrometers as exemplified by UV-2450PC from Shimadzu and light scattering instruments exemplified by the Zetasizer Nano™ from Malvern instruments, Raman spectrometers exemplified by the InVia™ spectrometer from Renishaw plc and circular dichroism (CD) spectrometers exemplified by the J-815 spectrometer from Jasco and the Chirascan™ spectrometer from Applied Photophysics. Generally the instruments described above can only record a single type of optical analytical data from a single sample. The CD spectrometers from JASCO and Applied Photophysics can, however, be modified to also acquire optical absorption and basic fluorescence intensity measurements although, again, these are only suitable for single sample cuvettes. Motorized sample changers are available for some instruments which typically hold up to 6 individual cuvettes.
Spectrometers and that perform a single type of measurement sequentially on multiple samples in SBS standard micro-titre plates in an automated fashion are also available. These are exemplified by the Spectramax™ 190 and the Spectramax Gemini™ spectrometer from Molecular Devices.
Instruments are available that are compatible with the SBS standard micro-titre plates described above that enable both fluorescence and optical absorption measurements to be made in the same instrument. Examples include the Infinte™ 200 from Tecan. Typically these illuminate and collect from either above or below the samples which presents various problems for the different measurement types. For example, problems occur in optical absorption measurements due to meniscus effects and path length dependence on the sample volume. Problems increase as the sample volumes are reduced.
A disposable multi-cuvette rotor is disclosed in U.S. Pat. No. 4,226,531 in which the sample cells are arranged in a radial configuration with the cells lying at the circumference of the disc. Although in this configuration, the cells can have three optical windows, potentially allowing both optical absorption and fluorescence or light scattering measurements to be made, no such applications are described and indeed the device as described would not lend itself to being used to perform simultaneous multiple optical measurements. Not only does the radial configuration of cells limit the total number of cells which can be accommodated in a given sample plate but it also limits optical access to only three optical windows. Moreover, as the liquid sample does not contact all three optical windows, optical absorption measurements are therefore potentially subject to errors due to differences in sample depth and meniscus effects.
In a number of important applications, such as protein thermal stability measurements, samples are available in only very small quantities and are not re-usable between tests which can prevent the separate application of multiple optical measurements. It would therefore be advantageous if multiple types of optical analysis could be performed simultaneously on small sample volumes. In addition simultaneously performing multiple analytical measurements from the same sample volume could potentially speed up measurements from large numbers of samples in applications such as high throughput screening.
As sample volumes decrease it becomes increasingly important to collect scattered, emitted or transmitted light from the sample with as high efficiency as possible and this is best achieved by using high numerical aperture optics. However, existing arrayed sample containers such as the widely used 96-well plates, restrict optical access to the sample volume, impairing the efficiency of the light collection.
Each of the different optical analysis methods has its own optimum geometric configuration for illumination and collection of the light in which can the signal to noise ratio of the acquired data is improved. As sample volumes are reduced the need to optimise the optical configuration becomes increasingly important since the amount of desirable signal decreases with sample volume whilst unwanted interference due to scattering or auto-fluorescence from the container increases. A sample container that allows the optimum optical configuration to be simultaneously maintained for multiple analytical methods would therefore be advantageous.
In addition in many applications it is desirable to be able to sequentially analyse multiple samples in an automated fashion in order that more experiments may be performed with in less time and with less dependence on human expertise.
Another common requirement for many biochemical and other applications is the facility to control the temperature of samples as they are analysed. It would therefore be desirable for any array of sample containers to be compatible with efficient heating and cooling of the sample during the application of multiple optical analytical techniques.
Moreover, in many applications it is desirable to have sample containers that are cheap enough to be disposable due to the cost and difficulty of effective cleaning.
As is apparent from the above discussion, there therefore remains a continuing need for the development of an improved system for obtaining multi-modal optical measurements from multiple samples, in particular which allows for the analysis to be carried out rapidly and automatically, suitably in high through-put manner.